CN110923294A - STD nucleic acid extraction and detection reagent and method - Google Patents

STD nucleic acid extraction and detection reagent and method Download PDF

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CN110923294A
CN110923294A CN201911351865.1A CN201911351865A CN110923294A CN 110923294 A CN110923294 A CN 110923294A CN 201911351865 A CN201911351865 A CN 201911351865A CN 110923294 A CN110923294 A CN 110923294A
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解信美
李红东
苗保刚
李明
彭年才
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SUZHOU TIANLONG BIOTECHNOLOGY CO Ltd
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Abstract

The invention discloses an STD nucleic acid extraction and detection reagent and a method, wherein the reagent comprises a nucleic acid extraction reagent and a nucleic acid detection reagent, the nucleic acid extraction reagent comprises cell lysate, nucleic acid washing solution and nucleic acid eluent, the nucleic acid detection reagent comprises a freeze-drying multiplex PCR reaction buffer solution, specific primers and probes of STD different pathogen target genes, internal standard quality control specific primers and probes and the like, and the STD different pathogens comprise chlamydia trachomatis, gonococcus, ureaplasma urealyticum and ureaplasma parvum. The invention provides a reagent for extracting, purifying and detecting and typing STD nucleic acid, which has the advantages of high sensitivity, simple and convenient use and operation, short time consumption and low professional requirement on operators, and the whole packaged reagent can be transported and stored at normal temperature.

Description

STD nucleic acid extraction and detection reagent and method
Technical Field
The invention relates to the field of nucleic acid extraction and purification, in particular to a reagent and a method for extracting and detecting STD nucleic acid.
Background
Common pathogens of Sexually Transmitted Diseases (STDs) are the following: chlamydia Trachomatis (CT), gonococcus (NG), Ureaplasma Urealyticum (UU), Ureaplasma Parvum (UP).
Among the conventional pathogen detection technologies, there are: (1) and a separation culture technology: pathogen isolation culture, the gold standard for early pathogen detection. The method has high specificity and sensitivity, but the clinical detection time is too long, the process is complicated, and the method is not suitable for large-range detection. (2) And immunological detection: pathogens are detected from protein levels in an antigen-antibody based immune response. The method has the problems of low detection sensitivity, large influence of environment on specificity and the like, long detection window period, incapability of meeting diagnosis and treatment requirements, suitability for preliminary screening, incapability of serving as a basis for timely diagnosis, incapability of identifying different subtypes of the same pathogen and the like. (3) And a detection method based on PCR: the PCR method comprises common PCR, a fluorescence PCR method, a gene chip, a sequencing method and the like, wherein pathogens are detected from a nucleic acid level, and the whole experiment needs 1-2 hours to be completed. The main disadvantage of this method is that PCR detection is carried out by means of PCR instrument or expensive real-time quantitative PCR instrument, and other supporting equipment, special PCR laboratory and professional operator. The PCR detection cannot realize instant detection, bedside diagnosis and scene application without specific laboratory detection conditions, so that the detection requirements of a basic level, a user terminal and a field cannot be met. Meanwhile, PCR detection may have the problems of low sensitivity, false negative caused by loss of toxic plasmids and the like.
At present, a multi-detection kit of pathogen CT/NG/UU/UP for sexually transmitting diseases based on a multi-PCR or multi-fluorescence PCR method generally selects only endogenous virulence plasmid genes of the pathogen for detecting CT and NG genes, and the plasmid is easy to lose, so that the missed detection of the pathogen is easy to cause. In addition, most of the kits can only detect ureaplasma urealyticum and cannot distinguish ureaplasma urealyticum from ureaplasma parvum. Furthermore, the use of these PCR detection reagents requires the extraction and purification of nucleic acids from a sample and the amplification and detection of the nucleic acid-extracted product using manual or semi-automatic nucleic acid extraction equipment. In addition, most of the detection kits need low-temperature storage and transportation, and the storage and transportation environment of the reagents is high. Moreover, the use of the reagent has high requirements on the professional skills and hardware equipment of operators, and is not favorable for clinical popularization.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an STD nucleic acid extraction and detection reagent and a method.
In order to achieve the purpose, the invention provides the following technical scheme: the reagent comprises a nucleic acid extraction reagent and a nucleic acid detection reagent, wherein the nucleic acid extraction reagent comprises cell lysate, a nucleic acid washing solution and a nucleic acid eluent, the nucleic acid detection reagent comprises a freeze-drying multiplex PCR reaction buffer solution, specific primers and probes of STD different pathogen target genes and specific primers and probes of an internal standard quality control gene, and the STD different pathogens comprise chlamydia trachomatis, gonococcus, ureaplasma urealyticum and ureaplasma parvum.
Preferably, the cell lysate comprises: 10-15 mM Tris-HCl, 2.5-5 mM EDTA, 30-40% TritonX-100 and an endoplasmic control plasmid template;
the nucleic acid washing solution comprises: 2.5-5 mM Tris-HCl, 0.1-0.2 mM EDTA, 0.7% NaCl;
the nucleic acid eluting solution comprises: 1.5 to 2.5mM Tris-HCl.
Preferably, the lyophilisable multiplex PCR reaction buffer comprises: 50-70 mM Tris-HCl, 15-30 mM KCl, 2.5-4.5 mM MgCl2, 20-40 μ g/μ L BSA, 0.2mM dATP, 0.4mM dUTP, 0.2mM dCTP, 0.2mM dGTP, 2-3U DNA polymerase, 0.1-0.2U TS-UNG enzyme, 0.3-0.5M trehalose, 0.001-0.002M PEG8000, 0.04-0.08M mannitol.
Preferably, the sequences of the specific primers and probes of the target genes of different STDs and the sequences of the specific primers and probes for internal standard quality control are as follows:
Figure BDA0002335192180000031
Figure BDA0002335192180000041
wherein, F is an upstream primer sequence, R is a downstream primer sequence, and P is a double-labeled probe sequence.
Preferably, the PCR detection reagent simultaneously detects 5 fluorescence detection channels, each of which detects an STD pathogen or internal control.
Preferably, the STD nucleic acid extraction and detection reagent is packaged in a closed reagent device, the reagent device comprises a nucleic acid extraction chamber and a nucleic acid detection chamber, and the nucleic acid extraction reagent and the nucleic acid detection reagent are packaged in the nucleic acid extraction chamber and the nucleic acid detection chamber, respectively.
The invention also discloses another technical scheme: an STD nucleic acid extraction and detection method comprising:
s1, collecting a sample to be detected of the STD infected person;
s2, injecting the sample to be detected into a nucleic acid extraction chamber, and respectively cracking, washing and eluting the sample to be detected by cell lysis solutions, nucleic acid washing solutions and nucleic acid eluents pre-packaged in different nucleic acid extraction chambers to obtain a nucleic acid extraction purification substance;
s3, after the nucleic acid extraction purification materials respectively flow into the nucleic acid detection chamber, dissolving the nucleic acid detection reagent preset in the nucleic acid detection chamber to perform PCR amplification and fluorescence detection of the nucleic acid extraction purification materials.
The invention has the beneficial effects that:
1. the invention provides a reagent for extracting, purifying and detecting and typing Sexually Transmitted Disease (STD) nucleic acid, which has high sensitivity, can achieve 50 copies/reaction of the detection sensitivity of pathogens, is simple and convenient to use and operate, consumes short time and has low professional requirements on operators, and in addition, a PCR amplification reagent is packaged in a reagent device in a freeze-drying mode, so that the whole reagent can be transported and stored at normal temperature.
2. Under the action of a matched instrument, the invention can complete the extraction, purification and detection processes of four pathogens of Sexually Transmitted Diseases (STD) in the same closed device, thereby greatly simplifying the detection and analysis process of the four pathogens of the Sexually Transmitted Diseases (STD) CT/NG/UU/UP.
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FIG. 1 is a schematic front view of a reagent device of the reagent pack of the present invention;
FIG. 2 is a diagram showing the result of positive detection in 5 channels of the nucleic acid detecting reagent of the present invention.
Reference numerals:
1. nucleic acid detection chamber, 2, nucleic acid detection reagent, 3, sample adding port, 4, first reagent chamber, 5, second reagent chamber, 6, product chamber, 7, nucleic acid adsorption chamber, 8, fourth reagent chamber, 9, third reagent chamber, 10, Chlamydia Trachomatis (CT), 11, gonococcus (NG), 12, Ureaplasma Urealyticum (UU), 13, ureaplasma micans (UP), 14, internal standard quality control (IC).
Detailed Description
The technical solution of the embodiment of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention.
The STD nucleic acid extraction and detection reagent and the method disclosed by the invention can complete the whole process of extraction, purification and detection of nucleic acids of 4 pathogens of Sexually Transmitted Diseases (STD) CT/NG/UU/UP in the same closed device, the detection reagent has high sensitivity, simple and convenient use and operation, short time consumption and low professional requirement on operators, and the whole packaged reagent can be transported and stored at normal temperature.
The invention discloses an STD nucleic acid extraction and detection reagent, which comprises: the nucleic acid extraction reagent can complete extraction and purification of STD nucleic acid, and comprises cell lysate, nucleic acid washing solution and nucleic acid eluent, and specifically, the implementation of the cell lysate can comprise: 10-15 mM Tris-HCl, 2.5-5 mM EDTA, 30-40% TritonX-100 and an endoplasmic control plasmid template. In this embodiment, the cell lysate has a content of 700 to 800uL, and includes: 15mM Tris-HCl (Tris is Tris hydroxymethyl aminomethane), 5mM EDTA (ethylene diamine tetraacetic acid), 30% Triton X-100 (polyethylene glycol octyl phenyl ether), and an inner quality control plasmid template, wherein the pH value of the Tris-HCl is preferably 8.3.
The nucleic acid washing solution may be carried out by: 2.5-5 mM Tris-HCl, 0.1-0.2 mM EDTA, 0.7% NaCl. In this example, the nucleic acid washing solution was 700uL, and its components included: 5mM Tris-HCl, 0.1mM EDTA, 0.7% NaCl, wherein the pH value of the Tris-HCl is preferably 8.0.
The nucleic acid eluting solution may be implemented by: 1.5 to 2.5mM Tris-HCl. In this example, the nucleic acid eluate was 300uL, and its components included: 1.5mM Tris-HCl, wherein the pH value of the Tris-HCl is preferably 8.3.
Preferably, the nucleic acid extraction reagent is in a liquid state, so that it can be stored at room temperature.
The nucleic acid detection reagent is based on a real-time fluorescence PCR (polymerase chain reaction) technology, and is used for respectively detecting and analyzing Chlamydia Trachomatis (CT), gonococcus (NG), Ureaplasma Urealyticum (UU) and ureaplasma micum micans (UP) of STD, and comprises multiple PCR reaction buffer solution (PCR buffer) required by multiple fluorescence PCR detection, DNA polymerase (Taq enzyme), specific primers and probes of target genes of different pathogens of STD, and specific primers and probes controlled by internal standard substances, wherein the different pathogens of STD comprise Chlamydia Trachomatis (CT), gonococcus (NG), Ureaplasma Urealyticum (UU) and ureaplasma micum micans (UP). Preferably, the nucleic acid detecting reagent is a freeze-dried PCR detecting reagent, which can also be stored at normal temperature. The freeze-dried reagent can be used for simultaneously detecting 5 fluorescence detection channels, comprises FAM, JOE, TAMRA, ROX and CY5, and can be used for detecting any pathogen or internal standard quality control in Chlamydia Trachomatis (CT), gonococcus (NG), Ureaplasma Urealyticum (UU) and Ureaplasma Parvum (UP).
In particular, the lyophilisable multiplex PCR reaction buffer may be implemented to include: 50-70 mM Tris-HCl, 15-30 mM KCl, 2.5-4.5 mM MgCl2, 20-40 μ g/μ L BSA, 0.2mM dATP, 0.4mM dUTP, 0.2mM dCTP, 0.2mM dGTP, 2-3U DNA polymerase, 0.1-0.2U TS-UNG enzyme, 0.3-0.5M trehalose, 0.001-0.002M PEG8000, 0.04-0.08M mannitol. In this example, the lyophilisable multiplex PCR reaction buffer comprises: 50mM Tris-HCl, 30mM KCl, 4mM MgCl2, 20. mu.g/. mu.L BSA, 0.2mM dATP, 0.4mM dUTP, 0.2mM dCTP, 0.2mM dGTP, 3U DNA polymerase, 0.2U TS-UNG enzyme, 0.3M trehalose, 0.002M PEG8000, 0.04M mannitol.
The sequences of specific primers and probes for detecting STD Chlamydia Trachomatis (CT), gonococcus (NG), Ureaplasma Urealyticum (UU) and Ureaplasma Parvum (UP) and the sequences of the specific primers and probes for internal standard quality control are shown in the following table:
Figure BDA0002335192180000071
Figure BDA0002335192180000081
Figure BDA0002335192180000091
wherein, F is an upstream primer sequence, R is a downstream primer sequence, and P is a double-labeled probe sequence.
In the above table, the amounts of the upstream and downstream primers and the probe in the nucleic acid detection reagent are respectively: no. 1333 nM, No.2333nM, No. 3167nM; no. 4233nM, No. 5233nM, No. 6117nM; no. 7167nM, No. 8167nM, No. 967nM; no. 10167nM, No. 11167nM, No. 1267nM; no. 13333nM, No. 14333nM, No. 15167nM; no. 16333nM, No. 17333nM, No. 18167nM; no. 19233nM, No. 20233nM, No. 21117nM.
Preferably, the STD nucleic acid extraction and detection reagent is pre-packaged in a closed reagent device (the specific structure of which can be found in the nucleic acid extraction, purification and detection device disclosed in chinese patent application No. 201710371949.6), which can complete the whole process of extraction, purification and detection of nucleic acids of 4 pathogens of Sexually Transmitted Diseases (STD) CT/NG/UU/UP in a closed system under the cooperation of a matched instrument, and can greatly simplify the processes of extraction, purification and detection of STD nucleic acids.
Referring to fig. 1, the device specifically includes a first reagent chamber 4, a second reagent chamber 5, a third reagent chamber 9, a fourth reagent chamber 8, a product chamber 6, a nucleic acid adsorption chamber 7, a nucleic acid detection chamber 1, and a sample detection port 3.
The cell lysis solution is pre-packaged in the first reagent chamber 4, nucleic acid washing solutions are packaged in the second reagent chamber 5 and the third reagent chamber 9, and nucleic acid eluent is pre-packaged in the fourth reagent chamber 8. The nucleic acid detection reagent is pre-packaged in the nucleic acid detection chamber 1.
The STD nucleic acid extraction and detection method disclosed by the invention comprises the following steps of:
and S1, collecting a sample to be detected of the STD infected person.
Wherein, the washing liquid of the urethra swab or the vagina swab washed by normal saline can be used as the sample to be detected.
S2, injecting the sample to be detected into the nucleic acid extraction chamber, and respectively cracking, washing and eluting the sample to be detected by cell lysis buffer, nucleic acid washing solution and nucleic acid eluent which are pre-packaged in different nucleic acid extraction chambers to obtain the nucleic acid extraction purification product.
Specifically, 100-500 μ l (the sample volume can be increased or decreased according to the sample attribute, the demand of the extracted product, and the downstream experiment demand) of the sample to be detected is taken, the sample is injected into the first reagent chamber 4 through the sample injection port 3 of the reagent device, the cell lysate is packaged in the first reagent chamber 4, the added sample to be detected is subjected to full lysis treatment under the action of a matched instrument, the lysed mixed solution flows into the nucleic acid adsorption chamber 7 under the action of centrifugal force, the nucleic acid is captured in the chamber, and the waste liquid flows into a waste liquid cavity (the back of the reagent device, not shown in fig. 1).
The nucleic acid washing solution in the second reagent chamber 5 flows into the nucleic acid adsorption chamber 7 to wash the nucleic acid, the washed nucleic acid is still retained in the nucleic acid adsorption chamber 7, and the waste liquid flows into the waste liquid chamber.
The nucleic acid washing solution in the third reagent chamber 9 flows into the nucleic acid adsorption chamber 7 to wash the nucleic acid, the nucleic acid is still retained in the nucleic acid adsorption chamber 7 after washing, and the waste liquid flows into the waste liquid chamber.
The nucleic acid eluent encapsulated in the fourth reagent chamber 8 flows into the nucleic acid adsorption chamber 7, and the adsorbed nucleic acid is eluted to become a nucleic acid extraction purification product, and flows into the product chamber 6 under the action of centrifugal force.
S3, after the nucleic acid extraction and purification material flows into the nucleic acid detection chamber, the nucleic acid detection reagent pre-arranged in the nucleic acid detection chamber is dissolved to carry out PCR amplification and fluorescence detection on the nucleic acid extraction and purification material.
Specifically, the nucleic acid extraction purification product in the product chamber 6 is rotated back and forth to be fully mixed under the action of external force provided by a matched instrument, then flows into the nucleic acid detection chamber 1, the nucleic acid extraction purification product flowing into the nucleic acid detection chamber 1 dissolves the nucleic acid detection reagent 2 therein to form a complete PCR reaction system, and subsequent PCR amplification and fluorescence detection are performed under the action of the matched instrument. FIG. 2 is a diagram showing the results of 5-channel positive detection of nucleic acid detection reagents: curve 10 represents Chlamydia Trachomatis (CT), curve 11 represents gonococci (NG), curve 12 represents Ureaplasma Urealyticum (UU), curve 13 represents Ureaplasma Parvum (UP), and curve 14 represents internal quality control (IC).
In the whole detection process, only an experimental operator needs to add a sample into the reagent device, the whole processes of nucleic acid extraction, purification and PCR fluorescence detection of the sample are automatically carried out in a matched instrument, and the whole process of extraction, purification and detection of the nucleic acid of CT/NG/UU/UP 4 pathogens of the STD can be simplified.
The invention is based on multiplex fluorescence PCR detection technology, respectively carries out the design of primer probes for multiple pathogens of STD to be detected and added internal standard quality control, develops multiplex fluorescence PCR reagents of 5 fluorescence channels, then combines a closed nucleic acid extraction and purification device, pre-encapsulates the nucleic acid extraction reagent and the nucleic acid detection reagent in the device, can realize the whole process from the nucleic acid extraction and purification of CT, NG, UU and UP 4 pathogens of STD to the fluorescence PCR detection and analysis in a closed system under the cooperation of matched instruments, and can greatly simplify the process of the detection and analysis of CT, NG, UU and UP pathogens of STD. In addition, the nucleic acid extraction reagent in the embodiment is in a liquid state and can be stored at normal temperature, and the nucleic acid detection reagent is a freeze-dried reagent and can also be stored at normal temperature, so that the whole packaged reagent can be stored and transported at normal temperature.
Therefore, the scope of the present invention should not be limited to the disclosure of the embodiments, but includes various alternatives and modifications without departing from the scope of the present invention, which is defined by the claims of the present patent application.
GATATGTTCCGAGACGGAGCTT
TAGCTGCTTCCTTCTCTTCATGAG
5’FAM-ATAACTGCCTCCCTGCTTCCCAC-3’BHQ1
CTCAAGGACCAGCAAATAATCCTT
TCCGCATCCAAACCAATTGT
5’FAM-CCTGTCGCAGCCAAAATGACAGCTT-3’BHQ1
CCTGCCAGTCTTGGTCTGAAG
CGCTGACGTTTTCCGCTAA
5’ROX-CGCGCGGACAGTTGTTTTTCGACT-3’BHQ2
CGATTCCCCCGGATTTTC
AACTGGTTTCATCTGATTACTTTCCA
5’ROX-AGCGGCAGCATTCAATTTGTTCCGA-3’BHQ2
ATGAAGCACACAACAAAATGGCG
CAACGTGCATTTGCTTCAACTTC
5’JOE-AGTATGGGCGGGGGTTATGTATCTGG-3’BHQ1
AAGTAGCATATGATCAAGCTCATTCA
TGTTGTAATGATACAACGAGCATCAT
5’TAMRA-TCACCTGAAACATATCCACCACCCATACT-3’BHQ1
CTTCTTAGCCATCCCAAGATTCTC
GAAACGTCGGAATAGTTCAAATCA
5’CY5-CAGATTGAGAGAGATTCAGAGAGTCACATC-3’BHQ2

Claims (9)

1. The STD nucleic acid extraction and detection reagent is characterized by comprising a nucleic acid extraction reagent and a nucleic acid detection reagent, wherein the nucleic acid extraction reagent comprises cell lysate, a nucleic acid washing solution and a nucleic acid eluent, the nucleic acid detection reagent comprises a freeze-drying multiplex PCR reaction buffer solution, specific primers and probes of STD different pathogen target genes and specific primers and probes of an internal standard quality control gene, and the STD different pathogens comprise chlamydia trachomatis, gonococcus, ureaplasma urealyticum and ureaplasma parvum.
2. The STD nucleic acid extraction and detection reagent of claim 1,
the cell lysate comprises: 10-15 mM Tris-HCl, 2.5-5 mM EDTA, 30-40% TritonX-100 and an endoplasmic control plasmid template;
the nucleic acid washing solution comprises: 2.5-5 mM Tris-HCl, 0.1-0.2 mM EDTA, 0.7% NaCl;
the nucleic acid eluting solution comprises: 1.5 to 2.5mM Tris-HCl.
3. The STD nucleic acid extraction and detection reagent of claim 1,
the lyophilisable multiplex PCR reaction buffer comprises: 50-70 mM Tris-HCl, 15-30 mM KCl, 2.5-4.5 mM MgCl2, 20-40 μ g/μ L BSA, 0.2mM dATP, 0.4mM dUTP, 0.2mM dCTP, 0.2mM dGTP, 2-3U DNA polymerase, 0.1-0.2U TS-UNG enzyme, 0.3-0.5M trehalose, 0.001-0.002M PEG8000, 0.04-0.08M mannitol.
4. The STD nucleic acid extraction and detection reagent of claim 1, wherein the sequences of the specific primers and probes of STD different pathogen target genes and the sequences of the internal standard quality control specific primers and probes are as follows:
Figure FDA0002335192170000011
Figure FDA0002335192170000021
Figure FDA0002335192170000031
wherein, F is an upstream primer sequence, R is a downstream primer sequence, and P is a double-labeled probe sequence.
5. The STD nucleic acid extraction and detection reagent of claim 5, wherein the PCR detection reagent simultaneously detects 5 fluorescent detection channels, each of which detects an STD pathogen or internal control.
6. The STD nucleic acid extraction and detection reagent of claim 1, wherein the STD nucleic acid extraction and detection reagent is packaged in a closed reagent device comprising a nucleic acid extraction chamber and a nucleic acid detection chamber, the nucleic acid extraction reagent and the nucleic acid detection reagent being packaged in the nucleic acid extraction chamber and the nucleic acid detection chamber, respectively.
7. A STD nucleic acid extraction and detection method, comprising:
s1, collecting a sample to be detected of the STD infected person;
s2, injecting the sample to be detected into a nucleic acid extraction chamber, and respectively cracking, washing and eluting the sample to be detected by cell lysis solutions, nucleic acid washing solutions and nucleic acid eluents pre-packaged in different nucleic acid extraction chambers to obtain a nucleic acid extraction purification substance;
s3, after the nucleic acid extraction and purification product flows into the nucleic acid detection chamber, the nucleic acid detection reagent preset in the nucleic acid detection chamber is dissolved, so that PCR amplification and fluorescence detection of the nucleic acid extraction and purification product are carried out.
8. The STD nucleic acid extraction and detection method according to claim 7,
the cell lysate comprises: 10-15 mM Tris-HCl, 2.5-5 mM EDTA, 30-40% TritonX-100 and an endoplasmic control plasmid template;
the nucleic acid washing solution comprises: 2.5-5 mM Tris-HCl, 0.1-0.2 mM EDTA, 0.7% NaCl;
the nucleic acid eluting solution comprises: 1.5 to 2.5mM Tris-HCl.
9. The STD nucleic acid extraction and detection method according to claim 7,
the lyophilisable multiplex PCR reaction buffer comprises: 50-70 mM Tris-HCl, 15-30 mM KCl, 2.5-4.5 mM MgCl2, 20-40 μ g/μ L BSA, 0.2mM dATP, 0.4mM dUTP, 0.2mM dCTP, 0.2mM dGTP, 2-3U DNA polymerase, 0.1-0.2U TS-UNG enzyme, 0.3-0.5M trehalose, 0.001-0.002M PEG8000, 0.04-0.08M mannitol.
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